Natural (e.g., Cyclosporine, Vancomycin, Rapamycin) and synthetic (e.g., IXEMPRA®) macrocycles (Nature Reviews, 7:608-624 (2008)) incorporating either peptidic or non-peptidic constituents have found successful utility as therapeutics in treating many human ailments ranging from cancer, infectious diseases, neuroscience, cardiovascular and immunological disorders. In spite of the success, many life threatening diseases remain untreatable. Accordingly, it would be desirable to develop novel synthetic methods for macrocyclization that facilitate access to novel macrocycles embedded with peptidic or non-peptidic scaffolds, with improved metabolic/pharmacokinetic and cell permeability properties, and with potential for activity towards novel biological disease targets of the future (Molecular Diversity, 9:171-186 (2005)). In this vein over the years synthetic methodologies towards constructing macrocycles have evolved beyond the classical lactam and lactone forming reactions to an armamentarium of methodologies that include, to mention a few, ring closing metathesis (RCM) reaction (Angew. Chem. Int. Ed., 37:3281-3284 (1998)), “click” chemistry (Drug Discovery Today, 8:1128 (2003)), SnAr reactions (J. Am. Chem. Soc., 71:8954-8956 (2006)), and cycloaddition reactions (J. Am. Chem. Soc., 127:3473-3485 (2005); Chem. Soc. Rev., 36:1674-1689 (2007)).
One of the challenges in specifically designing and synthesizing therapeutically useful macrocycles that inhibit protein-protein interactions central to many novel disease targets is the requirement of high-molecular weight molecules. Added accompanying prerequisite for such molecules is stereochemical and structural diversity in architecture such that they can adopt bioactive conformations to interact with protein target(s) and or a protein complex spanning large interacting surface areas (such as p53-MDM2, BCl2, notch complex) and are also cell permeable, preferably endowed with oral bioavailability (Angew. Chem. Int. Ed., 49:1-5 (2010)), and stable under physiological conditions. To address this from a future drug discovery perspective, the macrocycles field in parallel has shifted to the development of technology platforms of macrocycle stabilized peptides that encompass stabilized α-helix macrocycle stabilized peptides. In this regard, a few important technology platforms have emerged in recent years with several publications and patents disclosing biologically active molecules that target specific protein-protein interactions (e.g., transcription factor notch complex; Nature, 462(12):182-188 (2009)). It is noteworthy some these emerging technology platforms utilize macrocyclic forming reactions such as RCM reaction and the cycloaddition reaction mentioned above.
For example, WO 2004/077062 discloses a process for linking and cyclizing peptides containing cysteine groups. A peptide containing at least two cysteine moieties is alkylated with a scaffold-containing thiophile until two cysteine moieties are captured on to the scaffold to yield a macrocyclic or macrocycle stabilized peptide. This technique mimics the most common form of cyclization (i.e., disulfide bond) found among naturally occurring peptides and proteins, but does not provide a convenient means of preparing other types of cyclic structures due to its highly specific reactivity towards thiols only.
More recent publications such as WO 2010/034026, WO 2010/083347, and WO 2010/011313 disclose methods of producing macrocycle stabilized peptides by functionalizing two distant amino acid moieties on a protein or a peptide with olefinic residues that undergo ring closing metathesis (RCM) reactions to form a hydrocarbon staple. In particular, WO 2010/011313 discloses methods of ligating a macrocycle stabilized peptide to another peptide or a larger protein.
WO 2010/033617 discloses a method of preparing a macrocycle stabilized peptide by reacting (photochemical) an alkene moiety on a functionalized amino acid with a tetrazole moiety on another amino acid to form a pyrazoline cross-linking moiety via a (3+2) cycloaddition reaction.
To date, the widely used RCM based platform has been successful in making biologically active stabilized α-helix staple peptides with cell permeability and resistant to protease degradation. However, each platform's technology has limitations and synthetic challenges due to the reagents, reaction conditions, and potential compatibility issue with the composition of the peptidic substrate. For example, with the ring closing metathesis (RCM) platform there are potential issues and pitfalls involved with cost of reagents, length of synthetic sequences, toxicity of reagent (e.g., ruthenium catalyst), non-compatibility with methionine/cysteine, and scale-up and purification (Organic Process Research & Development, 513-515 (2005)). In addition, the potential lack of stereoselectivity of the product EZ double bond geometry during the construction of larger macrocycles can result in a product mixture of double bond regioisomers (J. Org. Chem., 3863-3868 (2006)).
Consequently there exists a need for novel technologies towards building macrocycles and macrocycle stabilized peptides that potentially could offer versatility, simplicity, efficiency, cost advantages, and synthetic compatibility with peptides. Such a technology could be useful in preparing larger quantities of high molecular weight proteins (>60 amino acids) that contain stabilized α-helix macrocycle stabilized or stitched peptide segments. Thus a novel platform would offer excellent opportunities to construct biologically useful molecules (either agonists, antagonists, or with a new function) for novel therapeutic use in broad disease areas.